The disclosure relates, in general, to the design and construction of an optofluidic device and, more particularly, to an optical cytometer for cell counting.
Continuous manipulation and separation of microparticles, both biological and synthetic, is important for a wide range of applications in industry, biology, and medicine. Traditional techniques of particle manipulation rely on laminar flow or differences in either particle mobility or equilibrium position in a flow with a variety of externally applied forces. Recently, microfluidic systems have been shown to be very useful for particle handling with increased control and sensitivity. Systems have been demonstrated that use scale-dependent electromagnetic forces, microscale hydrodynamic effects, or deterministic physical interactions and filters. However, the precision of microfluidic systems based on deterministic interaction with walls or posts may be limited by disturbances from random interparticle contact and spacing, and mechanical systems are prone to clogging. Additionally, throughput for particle manipulation based on external forces has been limited because the time for forces to act decreases with increasing flow rate.
It has been recently demonstrated that inertial lift forces in laminar microfluidic systems can be used to focus randomly distributed particles continuously and at high rates to a single streamline. In one aspect, this process is primarily controlled by the ratio of particle size to channel size and the flow characteristics of the system, but can be independent of particle density. This simple and robust method requires no mechanical or electrical parts, making it desirable for a number of applications.
One application of particular interest is flow cytometry, which is a common method for the analysis of cells and particles in biomedical research and clinical diagnostics. Current flow cytometers are large, robust bench top instruments capable of measuring optical scattering and fluorescence of cells and particles at extremely high throughput (˜1000's of events per second). While these systems have decreased in size over time, they are still not amenable to mobile point of care diagnostic settings or rugged environments due to the size and sensitivity of the measurement optics.
Integration of microscale fluidic and optical technologies is a promising approach for reducing the size and complexity of optical cytometers. Current approaches to optofluidic integration typically employ optical waveguides to direct light to a microfluidic flow cell, with many different design approaches for both the optical and fluidic system. Most designs employ either optical fibers to directly deliver light to a flow cell or slab waveguiding structures that employ photolithographic materials to guide light to a fluid channel defined within the slab. While using optical fibers directly is an attractive method because of the cost, availability, and excellent properties of commercial fibers, it does not allow shaping of the excitation or scattered light. Accordingly, such systems struggle with the discrimination of particles based on scattering because contamination from the excitation source can quickly mask the smaller scattering signals.
Systems that employ slab waveguides are an attractive alternative to direct insertion of fibers into microfluidic structures. Slab waveguides are formed by sandwiching a patterned material with a high refractive index between two substrates with a lower index of refraction. These systems are attractive because of the precision of photolithographic pattering of surface features that act as waveguiding structures. Slab waveguides have been used to optimize excitation beam shape, steer excitation light with total internal reflection, and collect scattered light. These systems, however, typically involve complex construction methods, require photolithographic patterning for each device, and employ materials that can autofluoresce or degrade with ultra-violet exposure.
Therefore, what is needed is an easily manufactured, microfluidic device that is capable of (i) controlling the spatial distribution of particles, (ii) analyzing the particles, and (iii) supporting mobile point-of-care diagnostics. It would also be desirable to achieve these capabilities in a cost-effective manner to enable true point-of-care diagnostics, particularly, in situations where cost is a driving consideration, such as in developing nations.